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From 2011.igem.org

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Revision as of 12:15, 4 October 2011

Present proteins to the outer membrane

   We used OmpA-His tag-GFP to test the conditions for presenting protein to the outer membrane.
   The design allows double check whether the C-terminal moeity can be presented outside.

OmpA, GFPHis tag 1507bp confirmed by sequencing

2. pet15b OmpA-HIV cleavage site-sh3 OmpA-sh3 (920bp), add HIV cleavage site to 968bp. Confirmed by sequencing

3. pet15b pro-rich –mCherry confirmed by sequencing

4. pet15b OmpA-HIV protease 1045bp confirmed by sequencing

2. Protein expression test 1. Pro rich-mCherry a. Test for proper inducing condition(table2) Table 2 group 1 2 Volume 3ml IPTG(mM) 0 0.1 0.5 1 Temperature 30℃ 18℃ Duration 5h 13h description Medium remained yellow. E.coli white Medium turned red E.coli cherry pink

After Pro-rich mCherry expression, significant color change can be observed (bacteria cells turned cherry red as a result of mCherry expression)

1-a b. SDSPAGE(Coomassie brilliant blue stain) No significant specific band observed

 1  2    3   4   5   6    7   8 Marker 9   10  11  12  13  14 
                           1-b 

c. Ni column purification

Elution Description 5mM Flowthrough still red, no color change within column 20mM洗脱 Flowthrough still red 200mM洗脱 Flowthrough red, significant color change within column can be observed(dark red to grey) Coomassie brilliant blue stain after purification

 1    2    3    4    5      6  Marker  7   8    9    10

1-c d. Western Blotting（1-d）

图1-d 1. OmpA-GFP membrane transportation a. Test for proper inducing condition(table4） Table 4 Group 1 2 Volumn 3ml IPTG (mM) 0 0.1 0.5 1 Temperature 30℃ 18℃ Duration 7h 12h Description Medium remained yellow. E.coli white Medium slightly changed to green E.coli Green florescence Florescence : green color can be observed by eyes b. Membrane fraction isolation 13000rpm, 30min. Supernatant: soluble protein and membrane fraction Pellet: non-sonicated cell, cell debris, non-soluble protein 51000rpm 1h Supernatant: soluble protein Pellet: Membrane fraction c. Western Blotting

                  13000rpm                51000rpm     
              S            P           S           P